| Glutathione (γ-glutamyl-L-cysteinylglycine, GSH) is the most abundant free thiol compound in cells of animals, plants and microorganisms. GSH acts as the principal redox buffer, plays an important role in oxidative stress response, and influences several essential processes such as gene expression, cell proliferation and apoptosis. Therefore, nowadays GSH finds wide applications in pharmaceutical, food and cosmetic industries, and the commercial demand for GSH is expanding.GSH is synthesized by the consecutive action ofγ-glutamylcysteine synthetase (GSH I, EC 6.3.2.2) and GSH synthetase (GSH II, EC 6.3.2.3) with the consumption of ATP. Compared with the fermentative method, the enzymatic production of GSH can achieve a higher concentration and be more beneficial to its final purification. However, to date, the enzymatic production of GSH has not been commercialized. On one hand, the requirement for ATP (2 M ATP for 1 M GSH) in GSH biosynthesis makes this process difficult to scale-up, for it is impractical from an economic point of view to add ATP directly on an industrial scale. Furthermore, high concentration of ATP and its metabolites such as ADP also inhibit the activity of GSH synthetase. On the other hand, a great deal of GSH I and GSH II with high catalytic activity are also essential to the enzymatic synthesis of GSH. In this study, a coupled system was constructed with E. coliΔadd/ade/pepT (pBV03) and S. cerevisiae WSH2. In this coupled system, ATP regeneration reaction was established with Ado as subtrate. Therefore, the GSH production increased with the enhancement of ATP regenerating-efficiency. The mechanism of ATP regeneration was also investigated in the coupled system. Furthermore, the key enzymes realated to GSH degradation were determined, and GSH degradation in E. coli was eliminated by the gene knockout and the optimization of culture condition. At last, a method, which was easy implement and free from significant adverse effects, are established for the enzymatic production of GSH. The main results were described as follows:(1) An adenosine deaminase (ADA, add)-deficiency recombinant E. coli JW1615 was constructed by disrupting add gene of E. coli BW25113. The results suggested that the knockout of add could block the transformation of Ado into Ino, and then decrease the generation of Hx from ATP. As a sequence, ATP was mainly transformed into Ade and Ado rather than Hx by E. coli JW1615 (pBV03). Moreover, both Ado and Ade could be used to regenerate ATP by S. cerevisiae WSH2. Therefore, GSH production was improved in the coupled system constructed with ADA-deficient E. coli JW1615 (pBV03) and S. cerevisiae WSH2. It reached 7.82 mM, which is 2.94-fold to the control coupled system constructed with E. coli BW25113 (pBV03) and S. cerevisiae WSH2. To generate 7.82 mM GSH, at least 15.64 mM ATP was required, which was more than the theoretical value of ATP generated from the initial 10 mM Ado. It suggests that ATP regeneration reaction was achieved in the coupled system between E. coli JW1615 (pBV03) and S. cerevisiae WSH2. However, small amount of Hx was still detected in Ado metabolism by E. coli JW1615 and GSH production by E. coli JW1615 (pBV03) with the addition of ATP. There are two pathways which transform Ado into Hx in E. coli. One pathway is from Ado to Hx via Ino, and the other is via Ade. These results suggested that the parallel pathway from Ado to Hx via Ade opened when the pathway from Ado to Hx via Ino blocked, otherwise, the pathway was almost closed. The transformation of ATP into Hx was mainly via the pathway from Ado to Ino and Hx. However, the transformation of Hx from ATP can not be completely blocked by knockouting add gene only.(2) The knockout of add and ade in E. coli completely blocked the transformation from ATP to Hx via Ado and Ade. Moreover, the main metabolites of ATP were Ado and Ade, which could be both used to generate ATP by the glycolytic pathway of S. cerevisiae WSH2. As a result, the coupled system constructed with E. coliΔadd/ade (pBV03) and S. cerevisiae WSH2 produced 10.88 mM GSH within 6 h, which was 4.03-fold of the control coupled system (2.70 mM) composed of E. coli BW25113 (pBV03) and S. cerevisiae WSH2. Comparing with the coupled system composed of E. coli JW1615 (pBV03) and S. cerevisiae WSH2, its GSH production increased 39.1% due to the further increase of ATP-regenerating efficiency. Improving the efficiency of regeneration system can increase GSH production in the coupled system. These results suggested that low GSH production in the coupled system was not caused by the low efficiency of ATP utilization, but the low efficiency of ATP generation.(3) In E. coli, the effect of single gene knockout on GSH degradation was investigated by disrupting the gene of enzymes related to GSH degradation.γ-Glutamyltranspeptidase (GGT) was the key enzyme of GSH degradation, and the knockout of ggt gene can greatly reduce theγ-GGT activity. Furthermore, the production of GSH improved significantly due to the decrease of GSH degradation caused byγ-GGT. However, the knockout of ggt gene could not completely inhibit the degradation of GSH. Tripeptidase (PepT) was another key enzyme of GSH degradation. However, the knockout of glutathione S-transferase (GST) gene has no effect on the reduction of GSH degradation. There was no substrate which can be used to accept the sulfur atom of GSH in our biosynthetic system. Therefore, in this study GST is not the key enzyme related to GSH degradation. Interestingly, the activity ofγ-GGT in the whole-cell was affected greatly by the culture condition. Optimizing the culture condition can almost totally eliminate the action of GGT on GSH degradation. Therefore, the knockout of ggt gene was unnecessary in the biosynthesis system constructed in this study. However, the culture temperature had no effect on the activity of PepT, therefore, the knockout of pepT was essential to inhibit the degradation of GSH caused by PepT. GSH degradation was nearly completely inhibited in the biosynthetic system of GSH constructed with tripeptidase-deficient recombinant E. coli JW1113 (pepT -, pBV03), which was cultured at 30oC for 3 h and 42oC for 5 h.(4) A recombinant E. coli JW1617 was constructed by disrupting lpp gene of E. coli BW25113. The growth curves of E. coli JW1617 and E. coli BW25113 was investigated. The results suggested that the knockout of lpp gene had no effect on the growth of E. coli JW1617 while the rate and production of GSH synthesis by E. coli JW1667 was significantly increased compared to that of E. coli BW25113 (pBV03). After 1 h, GSH production of E. coli JW1667 reached 12.66 mM, which was 5.75-fold of E. coli BW25113 (pBV03). These results suggested that the knockout of lpp gene increased the production of GSH enzymatic production by the enhancement of cell permeability. Compared with the direct addition of toluene, the effect of lpp gene knockout on the recycle times of GSH production by E. coli was investigated. GSH production of two permeabilizing methods was almost the same at the first synthesis, and then reduced with the increase of recycle times. At the fifth recycle, GSH production of the direct addition of toluene reduced 22.1%, while that of lpp gene knockout reduced only 11.0%. These results suggested that lpp gene knockout could be a new permeabilizing method for the cell permeabilization of E. coli, which was easy to achieve and free from significant adverse effects of current permeabilizing methods.(5) ATP metabolites of the coupled system composed of E. coliΔadd/ade/pepT (pBV03) and S. cerevisiae WSH2 was determined. The result suggested that the irreversible conversion of ATP into Hx was still existed in this coupled system. As described in previous study, the convesion of ATP was completed blocked in E. coliΔadd/ade/pepT (pBV03), therefore, the metabolites of Ado and Ade was determined. After 2 h, almost 88% of Ado was used for the synthesis of ATP by S. cerevisiae WSH2 while Ino, Hx and Ade could not be determined. This result suggested that the adenosine deaminase reported in KEGG did not work. Furthermore, the conversion of Ado into Ade did not happen in S. cerevisiae WSH2 due to Ade could not be determined in the Ado metabolism. However, a great deal of Hx (1.97 mM) and Ino (0.67 mM) was detected in Ade metabolism. Therefore, the production of Hx was associated with the action of adenine deaminase. Inhibiting the activity of adenine deaminase significantly reduced the conversion from Ade to Hx, and increased the efficiency of ATP regeneration and GSH production. Postponing the addition of cells of E. coliΔadd/ade/pepT (pBV03) could also increase GSH production of the coupled system. On one hand, the added Ado was whole used to regenerate ATP by S. cerevisiae WSH2 after postponing the addition of E. coli cells. On the other hand, the irreversible transformation from adenosine (Ado) to hypoxathine (Hx) in E. coliΔadd/ade/pepT (pBV03) was completely blocked by the disruption of add and ade, thus, more Ado was used to regenerate ATP by S. cerevisiae WSH2. At last, ATP-regenerating efficiency was improved, and GSH production reached to 18.32 mM, which was 6.89-fold of the control.
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